Field of the Invention
Immunoassays of antigenic substances (antigens) present in biologic fluids are often used in the evaluation of physiologic or pathologic states in animals including humans. Components of immunoassays are the antigen and specific antibody, and the assay involves the binding of these components to each other in a way which is dependent on the concentration of each component.
An antigen has two properties: (1) the capacity to stimulate the formation of corresponding antibodies and (2) the ability to react specifically with the corresponding antibodies. Essentially all proteins, many polysaccharides, nucleoproteins, lipoproteins, synthetic polypeptides and smaller molecules, such as haptens, if linked to larger proteins, behave as antigens. A hapten is a small, incomplete antigen being incapable alone of causing the production of antibodies but capable of reacting with specific antibodies which are produced when the hapten, attached to a larger antigenic substance, is injected into an animal. Induction of antibody formation or immunization occurs when the antigen is introduced into a living body through the skin, muscle, intraperitoneal injection or intravenous injection. The ability of an antigen to stimulate antibody production is enhanced if the compound is retained in the tissues. Because of this finding, materials which allow for the slow release of the antigen for prolonged periods, also known as adjuvants, are used to increase the antibody response to antigen.
The ability to elicit an antibody response is dependent upon the biological system and conditions employed, dose, route of administration, etc. In response to an antigen, the animal produces antibodies which are capable of reacting to each and every site of the antigen it recognizes as foreign. The smallest portion of the antigen that can determine an antibody specificity is termed an antigenic determinant or epitope. The many antibodies produced against a given antigen is a result of a corresponding large number of different antibody-producing cell lines and are termed polyclonal antibodies or polyclonal antisera. Polyclonal antibodies are mixtures of many different antibody specificities against all the many antigens an animal has been exposed to in a lifetime. Because most of the antibodies present in antisera are not specific for any given antigen, there are difficulties associated with purifying antibodies specific for a given antigen. Polyclonal antibodies are extremely heterogeneous with respect to their affinities for antigen; the effects of temperature, pH and ionic strength on rates of immunoreaction and variability of the immune response to antigen from animal to animal. The use of monoclonal antibodies in the present invention, obtained by a process described by Kohler and Milstein in Nature 256 495-497, 1975, eliminates many problems associated with the heterogeneous nature of polyclonal antibodies and provides a homogeneous reagent that has a uniform binding affinity and specificity. A monoclonal antibody reacts with only a very small portion of the larger, more complex antigen called an antigenic determinant or epitope.
The presence of antigen in a biologic fluid can be estimated by comparing the reaction of the unknown sample to that of the same or similar antigen. By using standard quantities of antigen or standards in an immunoassay, the concentration of an unknown amount of antigen can be estimated by a process known as standardization. The reaction of the standards relate to their content of antigen and can be graphed on the Y-axis while their known antigen content can be graphed on the X-axis to produce a standard curve. The reaction of an unknown is then found on the Y-axis and used to fix a value for the unknown on the X-axis. This process of standardization relies on the similarity of reactivity of the standards added to the assay and the unknown antigen.
Standard antigens may be altered in their reactivity with a given antibody over time and therefore it is necessary to monitor or control the standardization process so that results of assays run at different times can be compared. This process of controlling immunoassays involves the assay of control samples which contain the antigen in a fluid similar to that of the test samples being measured. The control samples are assayed every time the assay is standardized and are assumed to react in a similar fashion over the course of time. Variability and trends in the assayed control values reflects differences in the standard curve which may occur due to deterioration of the standards. It is important to have stable materials for standardization and control of immunoassays to assure the reproducibility and accuracy of the immunoassay. Accuracy refers to the assessment of how close a value is to the true value while precision refers to the degree of spread of a series of observations or reproducibility where the spread is specifically stated, that is, within run precision, day-to-day precision, etc. These terms are discussed in detail by Theodore Colton in his book, Statistics in Medicine, Little, Brown and Company Publishers, p. 38 (1974). Controls and standards, therefore, become as important to the validity of any immunoassay as the antibody and the antigen.
Certain immunoassays are particularly difficult to standardize and control because the antigen of interest is unstable and does not show uniform reactivity to antibodies. Minor alterations of certain antigens as would be necessary for production of stable standards and controls may significantly alter the binding of antibody which is essential for immunoassay. Even more subtle changes may occur in the structure of antigens with respect to their tertiary or quaternary structure adversely affecting antibody-antigen reactions. A problem, therefore, exists in the preparation of controls and standards for certain immunoassays as it is necessary to store standards and particularly controls over extensive periods of time in order to be useful for the control of the accuracy and precision of the assay. The standards and controls must also be preserved in such a way that they maintain uniform reactivity with the antibody. This invention relates to a process whereby stable controls and standards are prepared which react uniformly with antibody and provide for the long-term standardization and control of immunoassays involving labile antigens.
One important aspect of this invention involves the isolation and purification of specific epitopes for use as standards and controls in immunoassays involving monoclonal antibodies. Monoclonal antibodies are specific for epitopes which are normally present only once, but can be repetitive on certain antigens. Epitopes of proteins may be as small as 5 to 7 amino acid residues and may represent a specific linear amino acid sequence or a spatial arrangement of amino acids dependent upon conformation. Using a monoclonal antibody of suitable affinity the corresponding epitope may be isolated and purified from a mixture of antigens or from modified or fragmented antigen. Generally, the larger the antigen, the more likely it is to be unstable and degraded into fragments or smaller pieces. Since epitopes are always smaller than the intact antigen, epitope stability is invariably greater than the stability of the native antigen. While polyclonal antibodies may be greatly affected by the breakdown of the native antigen, carefully selected monoclonal antibodies, particularly those reactive with a specific amino acid sequence, may remain unaffected because the epitope being defined by the monoclonal antibody resides completely within a fragment of the native antigen. An important aspect of this invention is selecting monoclonal antibodies whose epitopes are not destroyed after fragmentation of the native antigen.
The binding of substances to surfaces enhances the stability of that substance. Antigens and/or antibodies have been bound to solid surfaces previously in the prior art to facilitate the separation of antibody-bound antigen from free antigen following reaction. An important distinction in this invention is the binding of an antigen and/or epitope to particles so that the bound epitope is stabilized in such a way as to make it useful in the preparation of standards and controls for immunoassays. Additionally, the stabilization of epitopes on particles allows for a much larger surface area and a much higher concentration of epitope for subsequent reaction. This significantly reduces reaction time and makes kinetic assays practical.
There is a well recognized relationship between the amount of cholesterol in the blood and atherosclerosis or coronary artery disease as reported in the Complete Home Medical Guide, of the Columbia University College of Physicians and Surgeons, [Crown Publishers, page 367, 1985]. Since cholesterol is a fatty substance (lipid) that is not soluble in blood, which is mostly water (and fats and water do not mix), cholesterol must associate with a detergent-like substance before it can travel through the blood. One such substance is a protein designated an apolipoprotein, which, when combined with lipid, forms a molecule called a lipoprotein. There are different types of lipoproteins, which are often classified by their size or density as determined by highspeed centrifugation (ultracentrifugation). The heaviest is high-density lipoprotein (HDL), which has the highest proportion of protein. Low density lipoprotein (LDL) is lighter than HDL, and carries a larger proportion of cholesterol. Very low density lipoprotein (VLDL) carries the largest proportion of triglyceride, a lipid that is important in fat metabolism. Recent studies suggest that HDL carries cholesterol away from arterial cells and is therefore important in balancing the accumulation of cholesterol and other fats within arteries. LDL carries cholesterol to arterial cells and is believed to be a major factor in the development of atherosclerosis. Thus, in blood plasma a high level of HDL cholesterol in relationship to LDL cholesterol, is now considered desirable. The higher the ratio of HDL to LDL cholesterol, the better.
Recent evidence indicates that the quantity of the major apolipoproteins of HDL (apolipoprotein A-I, apo AI) and LDL (apolipoprotein B, apo B) or apolipoproteins A-I and B (apo A-I and apo B), respectively, are even better measures of risk of developing heart attack than the measurement of HDL and LDL cholesterol. There is a strong correlation (positive) with apo B (LDL) and an inverse correlation (negative) with apo A-I (HDL) with respect to risk of developing clinically manifest coronary heart disease. Further refinements of the methods of quantitative determination of the associated apolipoproteins is expected to lead to the identification of more precise relationships relating to the causation of heart attacks in humans.
A particular problem in immunoassays involving lipoprotein apolipoprotein is the number of reactions that are well known to occur which alter protein structure and conformation of apolipoprotein antigens which may alter immunoreaction. Such reactions include lipid peroxidation, protein fragmentation due to peroxides and oxygen, enzymatic fragmentation and aggregation. Any of these reactions may adversely affect the immunoassay of apo B and apo A-I. There is a critical need for an improvement in standardizing and controlling assays involving apo B and apo A-I because they are particularly labile. Alteration of their lipid environment is well known to change conformation and immunoreactivity. Accordingly, one important aspect of this invention is to provide particle-stabilized epitopes specific for apo B and apo A-I for use as standards and controls in immunoassays of these important components of LDL and HDL.